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Aus der medizinische Klinik und Poliklinik IV der
Ludwig-Maximilians-Universität München
Direktor: Prof. Dr. med. Martin Reincke
________________________________________
Preclinical in vitro investigation to evaluate
novel molecular targeted therapy options for
neuroendocrine neoplasms
________________________________________
Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München
vorgelegt von
Elke Tatjana Aristizabal Prada
aus München
2018
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Mit Genehmigung der Medizinischen Fakultät der
Universität München
Berichterstatter: Prof. Dr. med. Christoph J. Auernhammer
Mitberichterstatter: Priv.- Doz. Dr. med. Klaus Metzeler Prof. Dr. med. Thomas Knösel
Mitbetreuung durch den promovierten Mitarbeiter:
Dekan: Prof. Dr. Reinhard Hickel
Tag der mündlichen Prüfung:
05.07.2018
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Eidesstattliche Versicherung
Aristizabal Prada, Elke Tatjana
Name, Vorname
Ich erkläre hiermit an Eides statt,
dass ich die vorliegende Dissertation mit dem Thema
Preclinical in vitro investigation to evaluate novel molecular targeted therapy options for
neuroendocrine neoplasms
selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle
Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich
gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe.
Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher
Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde.
München, den 12.08.18 Elke Tatjana Aristizabal Prada
Ort, Datum Unterschrift Doktorandin/Doktorand
Eidesstattliche Versicherung Stand: 31.01.2013
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CONTENTS
1. Abbreviations………………………………………………………………………….…..1
2. Publications……………………………………………………………………………..…3
3. Introduction……………………………………………………………………………...…5
3.1. General introduction into neuroendocrine tumors…………………………………...5
3.2. The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in
dual-targeting approaches demonstrates antitumoral efficacy in neuroendocrine
tumors in vitro………………………………………………………………………………...6
3.3. The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in
combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor
cells……………………………………………………………………………………………8
4. Abstract…………………………………………………………………………………...11
5. Zusammenfassung………………………………………………………………………12
6. Publication 1……………………………………………………………………………...13
7. Publication 2……………………………………………………………………………...14
8. Acknowledgements……………………………………………………………………...15
9. References…………………………………………………………………..…………...16
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1. ABBREVIATIONS
Chk Checkpoint kinase
CDK Cyclin-dependent kinase
p21 Cyclin-dependent kinase inhibitor 1
p16 Cyclin dependent kinase inhibitor 2A
Caspase Cysteinyl-aspartate specific protease
DNA Deoxyribonucleic acid
MDM2 E3 ubiquitin ligase mouse double minute 2 homolog (MDM2)
EGFR Epithelial growth factor receptor
4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1
RAD001 Everolimus, mTORC1 inhibitor (Novartis, Basel)
ERK Extracellular signal-regulated kinase 1
FACS Flow cytometric analysis
5-FU 5-fluorouracil
γ-irradiation Gamma-irradiation
G1 phase Gap 1 phase
G2 phase Gap 2 phase
GEP-NET Gastroenteropancreatic neuroendocrine tumor
GTP Guanosine triphosphate
MTH1 Human Mut-T homologue 1
IC50 Inhibitory concentration of 50 %
IGFR Insulin-like growth factor receptor
mTOR Mechanistic / mammalian target of rapamycin
mTORC1 Mammalian target of rapamycin complex 1
µM Micro molar
MEK Mitogen-activated protein kinase kinase
TH588 MTH1 inhibitor (Thomas Helleday, Karolinska Institutet, Sweden)
nM Nano molar
NET Neuroendocrine tumor
% Percent
PI3K Phosphoinositid-3-kinase
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PARP Poly (ADP-ribose)-Polymerase
PCNA Proliferating-Cell-Nuclear-Antigen
Akt Protein kinase B
Raf Rapidly accelerated fibrosarcoma protein
Ras Rat sarcoma protein
ROS Reactive oxygen species
Rb Retino-blastoma protein
LEE011 Ribociclib, CDK4/6 inhibitor (Novartis, Basel)
p70S6K Ribosomal protein S6 kinase beta-1
S phase Synthesis phase
E2F Transcription factor E2F1
p53 Tumor suppressor p53
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2. PUBLICATIONS
Additive Anti-Tumor Effects of Lovastatin and Everolimus in vitro through
simultaneous Inhibition of Signaling Pathways; PLoS One. 2015 Dec;
doi: 10.1371/journal.pone.0143830. PMID: 26636335. Nölting S, Maurer J, Spöttl G,
Aristizabal Prada ET, Reuther C, Young K, Korbonits M, Göke B, Grossman A,
Auernhammer CJ.
Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells
with defective repair mechanisms. Int J Radiat Biol. 2016 Jun; doi:
10.3109/09553002.2016.1152412. PMID: 26967458. Prada Medina CA,
Aristizabal Tessmer ET, Quintero Ruiz N, Serment-Guerrero J, Fuentes JL.
The HDM2 (MDM2) Inhibitor NVP-CGM097 inhibits tumor cell proliferation and shows
additive effects with 5-fluorouracil on the p53 - p21 - Rb - E2F1 cascade in the
p53wildtype neuroendocrine tumor cell line GOT1; Neuroendocrinology. 2016 Nov; doi:
10.1159/000453369. PMID: 27871087. Reuther C, Heinzle V, Nölting S, Herterich S,
Hahner S, Halilovic E, Jeay S, Wuerthner JU, Aristizabal Prada ET, Spöttl G, Maurer
J, Auernhammer CJ.
The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in dual
-targeting approaches demonstrates antitumoral efficacy in neuroendocrine tumors in
vitro. Neuroendocrinology. 2017 Feb; doi: 10.1159/000463386. PMID: 28226315.
Aristizabal Prada ET, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in
combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor
cells. PLoS ONE. 2017 May; doi: 10.1371/journal.pone.0178375. PMID: 28542590.
Aristizabal Prada ET, Orth M, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for
neuroendocrine tumors: Results from multiple cell line models. PLoS ONE. 2017
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Aug; doi: 10.1371/journal.pone.0182852. PMID: 28800359. Nölting S, Rentsch J,
Freitag H, Detjen K, Briest F, Möbs M, Weissmann V, Siegmund B, Auernhammer
CJ, Aristizabal Prada ET, Lauseker M, Grossman A, Exner S, Fischer C,
Grötzinger C, Schrader J, Grabowski P.
GSK3α/β: A Novel Therapeutic Target for Neuroendocrine Tumors.
Neuroendocrinology. 2017 Oct; doi: 10.1159/000481887. PMID: 28968593.
Aristizabal Prada ET, Weis C, Orth M, Lauseker M, Spöttl G, Maurer J, Grabowski
P, Grossman A, Auernhammer CJ, Nölting S.
Old and New Concepts in the Molecular Pathogenesis of Primary Aldosteronism.
Hypertension. 2017 Oct; doi: 10.1161/HYPERTENSIONAHA.117.10111. PMID:
28974569. Aristizabal Prada ET, Burrello J, Reincke M, Williams TA.
Targeted therapy of gastroenteropancreatic neuroendocrine tumours: preclinical
strategies and future targets. Endocr Connect. 2018 Jan; doi: 10.1530/EC-17-0286.
PMID: 29146887. Aristizabal Prada ET, Auernhammer CJ.
Comparative Genomics and Transcriptome Profiling in Primary Aldosteronism. Int J
Mol Sci. 2018 Apr; doi: 10.3390/ijms19041124. PMID: 29642543. Aristizabal Prada
ET, Castellano I, Sušnik E, Yang Y, Meyer LS, Tetti M, Beuschlein F, Reincke M,
Williams TA.
Tropomyosin receptor kinase: a novel target in screened neuroendocrine tumors.
Endocr Relat Cancer. 2018 May; doi: 10.1530/ERC-17-0201. PMID: 29563190.
Aristizabal Prada ET, Heinzle V, Knösel T, Nölting S, Spöttl G, Maurer J, Spitzweg
C, Angele M, Schmidt N, Beuschlein F, Stalla GK, Blaser R, Kuhn KA, Auernhammer
CJ.
The role of GSK3 and its reversal with GSK3 antagonism in everolimus resistance.
Endocr Relat Cancer. 2018 Jun; doi: 10.1530/ERC-18-0159. PMID: 29895527.
Aristizabal Prada ET, Spöttl G, Maurer J, Lauseker M, Koziolek EJ, Schrader J,
Grossman A, Pacak K, Beuschlein F, Auernhammer CJ, Nölting S.
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3. INTRODUCTION
3. 1. General introduction into neuroendocrine tumors
Neuroendocrine tumors (NETs) of the gastroenteropancreatic (GEP) system are highly
complex and heterogeneous tumors, regarding their physiologic and genetic make-up
and originate from distinct cell precursors [1]. GEP-NETs are frequently metastasized
at the time of diagnosis, due to an absence of symptoms and curative resection is often
impossible [2-4]. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are
after colorectal cancer the second most common gastrointestinal malignancy [4] with
increasing incidence [5, 6]. Despite an increasing knowledge of the tumor biology,
genetics, epigenetics and novel biomarkers, the overall outcome and survival of GEP-
NETs has gained very little over the last decades [4, 7, 8]. Furthermore, systemic
therapeutic approaches for GEP-NETs, such as biotherapy, chemotherapy, molecular
targeted therapy and peptide receptor radionuclide therapy are limited in their
efficiency [9-11]. The paucity of successful therapeutic agents in GEP-NET is mostly
due to the complexity, the rarity, the intrinsic differences in malignant potential because
of their heterogeneity and the dissimilar clinical presentation [1, 4]. Molecularly
targeted therapies with the multi-tyrosine kinase inhibitor sunitinib or the mTORC1
inhibitor everolimus are approved for treatment of pancreatic NETs [12-14]. Everolimus
has recently also been approved for treatment of lung and gastrointestinal NETs [15].
The promising molecular targeting agent everolimus downregulates the mTOR /
p70S6K pathway, one of the most important and oncogenic signaling cascades in
GEP-NETs [16-18]. The PI3K-Akt-mTOR axis is frequently over-activated in cancers,
including GEP-NETs, causing cellular survival, proliferation and protein synthesis [11,
19]. Unfortunately only a subset of patients respond to everolimus treatment, due to
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intrinsic resistance or the development of a de novo resistance in response to long
term treatment [20-24]. Compensatory feed-back activation of PI3K-Akt signaling in
response to mTORC1 inhibition, as well as cross-talk activation of other signaling
pathways such as the Ras-Raf-MEK-ERK cascade in response to molecular targeting
help to escape cancer cell death and are well described in NET cells [19, 25-27].
Hence, novel therapeutic strategies, including molecularly targeted therapeutics, are
urgently needed [9, 14]. Dual-targeting approaches might provide a rationale to
overcome acquired or intrinsic resistance, prevent feed-backs within and between
signaling pathways and enhance single substance treatment [24, 28, 29].
The aim of this thesis was to evaluate different novel molecular targeted therapy
options for neuroendocrine neoplasms based on an in vitro model:
3.2. The novel cyclin-dependent kinase 4/6 inhibitor
ribociclib (LEE011) alone and in dual-targeting
approaches demonstrates antitumoral efficacy in
neuroendocrine tumors in vitro
Cyclin dependent kinases (CDKs) are the key regulators of the cell cycle and aberrant
expression of these CDKs, due to gene mutation, amplification or overexpression often
lead to the formation of cancer [30]. A very promising molecular targeting approach for
GEP-NETs is the selective CDK4/6 inhibitor LEE011 (Novartis, Basel) by
downregulating the proliferative CyclinD-CDK4/6-Rb axis (Fig. 1) [29]. Currently a
clinical phase 2 trial with LEE011 is recruiting patients with advanced NETs of foregut
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origin (NCT02420691) and another clinical phase 2 trial is recruiting patients with
CDK4/6 pathway activated tumors (NCT02187783), substantiating the probable
clinical relevance of targeting the CyclinD-CDK4/6-Rb axis.
Fig. 1. Proposed and simplified mode of action of the CDK4/6 inhibitor LEE011 on the cell cycle. a
Activated CyclinD-CDK4/6-Rb axis leads to G1/S cell cycle progression via the phosphorylation of Rb
and subsequent activation of the transcription factor E2F. b Blocking the CyclinD-CDK4/6-Rb axis leads
to G1 phase cell cycle arrest through either the endogenous CDK4/6 inhibitor p16 or the small molecule
CDK4/6 inhibitor LEE011
Using human neuroendocrine pancreatic BON1, pancreatic islet QGP1,
bronchopulmonary NCI-H727 and ileal GOT1 cell lines we demonstrated the
antitumoral efficacy of the novel cyclin-dependent kinase 4/6 inhibitor ribociclib
(LEE011) alone and in dual-targeting approaches in vitro [29]. In this study we applied
cellular survival assays, Western blot analysis, flow cytometric analysis (FACS) and
Caspase-3/7 activity assays. The cell viability decreased in a time- and dose-
dependent manner in BON1, QGP1 and NCI-H727 cells upon LEE011 treatment, while
GOT1 cells were treatment resistant. High expression levels of endogenous Rb and
Cyclin D1 were associated with treatment sensitivity towards CDK4/6 inhibition in
NETs. LEE011 caused dephosphorylation of Rb and a subsequent G1 phase cell cycle
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arrest. The combinational treatment of LEE011 with 5-fluorouracil (5-FU) or everolimus
showed a significant enhancement in inhibition of cell viability when compared to the
respective single substance treatments, due to PI3K-Akt-mTOR and Ras-Raf-MEK-
ERK pathway downregulation and cooperative cell cycle component downregulation.
Conversely, LEE011 also demonstrated antagonizing effects in combination with 5-
FU, by seemingly protecting NET cells from the DNA damaging effects of the
chemotherapeutic agent through blocking PARP cleavage and caspase 3/7 activity.
Taken together our results, CDK4/6 inhibition with LEE011 might be an efficient new
therapeutic rationale for NETs either alone or in dual-targeting approaches, especially
in combination with everolimus. We recommend clinical studies to ratify the efficacy of
LEE011 and everolimus as dual-targeted therapy in neuroendocrine tumors.
3.3. The MTH1 inhibitor TH588 demonstrates anti-tumoral
effects alone and in combination with everolimus, 5-FU
and gamma-irradiation in neuroendocrine tumor cells
Cancer cells often display a dysfunctional redox regulation with a high level of reactive
oxygen species damaging the DNA and the free nucleotide pool [31, 32]. The
nucleotide pool-sanitizing enzyme MTH1 (human Mut-T homologue 1) was shown to
be of pivotal importance for the progression and the survival of cancer cells, preventing
the incorporation of damaged nucleotides into the DNA (Fig. 2) [33, 34]. Recently,
several new small-molecule inhibitors targeting the nucleotide-sanitizing enzyme
MTH1 (TH287, TH588 and S-crizotinib) were described to specifically induce lethality
in a broad spectrum of cancer cells without harming untransformed tissues [35, 36].
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However the validity of MTH1 as a promising target against cancer has been
questioned recently and the striking anti-proliferative effects of MTH1 inhibitors such
as TH588 have been attributed to off-target effects [37-39].
Fig. 2. Proposed mode of action of TH588. MTH1 prevents the incorporation of damaged nucleotides
into the DNA and hence protects cancer cells from the lethal effects caused by reactive oxygen species
(ROS). By inhibiting MTH1 cancer cell death is restored.
With this study we contributed to the assessment of cellular mechanisms and
molecular signaling pathways implicated in the cellular response to TH588 treatment
[40]. Using a panel of heterogeneous NET cell lines (BON1, QGP1, H727 and GOT1),
we tested TH588 alone or in combination with the mTOR inhibitor everolimus, 5-FU
and -irradiation. Here we applied cellular survival assays, Western blot analysis,
Caspase-3/7 activity assays, FACS analysis, oxidative stress level assays and colony
formation assays to determine the clonogenic cell survival after -irradiation. TH588
MTH1 prevents DNA incorporation of G*and thus foments cancer cell survival, due to a gene pool sanitizing function. TH588 blocks MTH1 and restores cancer cell death.
ROS
dGTP
DNA incorporation of G* DNA damage cancer cell death
G*
Polymerase
MTH1
TH588
8-oxo-dGTP 8-oxo-dG = G*
8-oxo-dGMP
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caused cancer cell death by downregulating the PI3K-Akt-mTOR axis and inducing
apoptosis, rather than causing augmentation of the cellular oxidative stress level.
These effects were even enhanced when TH588 was combined with everolimus or 5-
FU, due to an even stronger downregulation of the PI3K-Akt-mTOR axis and
augmentation of apoptosis but not oxidative stress. Furthermore TH588 demonstrated
a radiosensitizing potential in combination with -irradiation (radiotherapy), one of most
important treatment modalities in nowadays cancer treatment [41]. Our data thus not
only provided new insights into how TH588 kills cancer cells but also depicted novel
perspectives for combinatorial treatment approaches encompassing TH588.
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4. ABSTRACT
GEP-NETs are highly complex, rare and heterogeneous tumor entities [1]. Current
treatment approaches are unsatisfying, due to their limited efficacy [2-4]. Ergo, novel
therapeutic strategies, including molecularly targeted therapeutics, are urgently
needed [9, 14]. The aim of this doctoral thesis was to evaluate different novel
molecular targeted therapy options for neuroendocrine neoplasms in vitro. By
targeting different signaling pathways, we obtained novel insights into NET tumor
biology and assessed possible novel treatment strategies. Several dual-targeting
approaches prevented feed-back loops within a signaling pathway, as well as cross-
talk activation between signaling pathways and enhanced single substance
treatment. This cumulative doctoral thesis is based on the following original
publications:
The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in dual -targeting approaches demonstrates antitumoral efficacy in neuroendocrine tumors in vitro; Neuroendocrinology. 2017 Feb; doi: 10.1159/000463386. PMID: 28226315. Aristizabal Prada ET, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in
combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS ONE. 2017 May; doi: 10.1371/journal.pone.0178375. PMID: 28542590. Aristizabal Prada ET, Orth M, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
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5. ZUSAMMENFASSUNG
GEP-NETs sind hoch komplexe, seltene und heterogene Tumor-Entitäten [1].
Derzeitige Behandlungsmöglichkeiten sind aufgrund ihrer begrenzten Effektivität
nicht zufriedenstellend [2-4]. Daher werden neue Behandlungsstrategien, wie
molekular zielgerichtete Therapien, dringend benötigt [9, 14]. Das Ziel dieser
Doktorarbeit war es, verschiedene neue molekular zielgerichtete Therapieansätze für
neuroendokrine Neoplasien in vitro zu evaluieren. Durch das „targeting“ von
verschiedenen Signalwegen konnten neue Einblicke in die Tumor-Biologie von NETs
erzielt und neue mögliche Behandlungsstrategien erstellt werden. Einige „duale-
targeting“ Ansätze konnten „feed-back“ Mechanismen innerhalb eines Signalwegs
oder die Aktivierung zwischen unterschiedlichen Signalwegen verhindern und
verbesserten zudem die Einzelsubstanzbehandlung. Die kumulative Dissertation
basiert auf den folgenden Originalveröffentlichungen:
The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in dual -targeting approaches demonstrates antitumoral efficacy in neuroendocrine tumors in vitro; Neuroendocrinology. 2017 Feb; doi: 10.1159/000463386. PMID: 28226315. Aristizabal Prada ET, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in
combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS ONE. 2017 May; doi: 10.1371/journal.pone.0178375. PMID: 28542590. Aristizabal Prada ET, Orth M, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
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6. PUBLICATION 1
The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in dual
-targeting approaches demonstrates antitumoral efficacy in neuroendocrine tumors in
vitro; Neuroendocrinology. 2017 Feb; doi: 10.1159/000463386. PMID: 28226315.
Aristizabal Prada ET, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
Link:
https://www.ncbi.nlm.nih.gov/pubmed/?term=The+novel+cyclin-
dependent+kinase+4%2F6+inhibitor+ribociclib+(LEE011)+alone+and+in+dual+-
targeting+approaches+demonstrates+antitumoral+efficacy+in+neuroendocrine+tumo
rs+in+vitro
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7. PUBLICATION 2
The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination
with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS
ONE. 2017 May; doi: 10.1371/journal.pone.0178375. PMID: 28542590. Aristizabal
Prada ET, Orth M, Nölting S, Spöttl G, Maurer J, Auernhammer CJ.
Link:
https://www.ncbi.nlm.nih.gov/pubmed/?term=The+MTH1+inhibitor+TH588+demonstr
ates+anti-tumoral+effects+alone+and+in+combination+with+everolimus%2C+5-
FU+and+gamma-irradiation+in+neuroendocrine+tumor+cells
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8. ACKNOWLEDGMENTS
Besonders danken möchte ich Prof. Dr. med. Christoph Auernhammer, Dr. med.
Svenja Nölting, Dr. Michael Orth, Gerald Spöttl, Julian Maurer und Nina Schmidt für
eine sehr schöne und produktive Zeit im Labor.
Besonderer Dank gilt auch meinen Eltern Gisela Aristizabal Prada, Evi Weikl-Ertl und
Eduardo Aristizabal Prada, meinem Ehemann Javier Mauricio Villamizar Cujar,
meinen Paten Margit Leonhardt, Dr. Gisela Lindemann und Konrad Kellermann
sowie meinen Freunden, die mich auf meinem Weg durch das Studium liebevoll
begleitet und unterstützt haben.
Ebenso möchte ich der FAZIT-Stiftung sehr herzlich danken für die finanzielle
Unterstützung während meiner Doktorarbeit
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9. REFERENCES
1 Schimmack S, Svejda B, Lawrence B, Kidd M, Modlin IM: The diversity and commonalities of gastroenteropancreatic neuroendocrine tumors. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie 2011;396:273-298. 2 Frilling A, Akerstrom G, Falconi M, Pavel M, Ramos J, Kidd M, Modlin IM: Neuroendocrine tumor disease: an evolving landscape. Endocrine-related cancer 2012;19:R163-185. 3 Modlin IM, Gustafsson BI, Moss SF, Pavel M, Tsolakis AV, Kidd M: Chromogranin A--biological function and clinical utility in neuro endocrine tumor disease. Annals of surgical oncology 2010;17:2427-2443. 4 Modlin IM, Oberg K, Chung DC, Jensen RT, de Herder WW, Thakker RV, Caplin M, Delle Fave G, Kaltsas GA, Krenning EP, Moss SF, Nilsson O, Rindi G, Salazar R, Ruszniewski P, Sundin A: Gastroenteropancreatic neuroendocrine tumours. The Lancet Oncology 2008;9:61-72. 5 Modlin IM, Moss SF, Chung DC, Jensen RT, Snyderwine E: Priorities for improving the management of gastroenteropancreatic neuroendocrine tumors. Journal of the National Cancer Institute 2008;100:1282-1289. 6 Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, Mares JE, Abdalla EK, Fleming JB, Vauthey JN, Rashid A, Evans DB: One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26:3063-3072. 7 Karpathakis A, Dibra H, Pipinikas C, Feber A, Morris T, Francis J, Oukrif D, Mandair D, Pericleous M, Mohmaduvesh M, Serra S, Ogunbiyi O, Novelli M, Luong T, Asa SL, Kulke M, Toumpanakis C, Meyer T, Caplin M, Beck S, Thirlwell C: Progressive epigenetic dysregulation in neuroendocrine tumour liver metastases. Endocrine-related cancer 2017;24:L21-l25. 8 Kidd M, Modlin I, Oberg K: Towards a new classification of gastroenteropancreatic neuroendocrine neoplasms. Nature reviews Clinical oncology 2016;13:691-705. 9 Auernhammer CJ, Goke B: Therapeutic strategies for advanced neuroendocrine carcinomas of jejunum/ileum and pancreatic origin. Gut 2011;60:1009-1021. 10 Cives M, Strosberg J: Radionuclide Therapy for Neuroendocrine Tumors. Current oncology reports 2017;19:9. 11 Pavel ME, Sers C: WOMEN IN CANCER THEMATIC REVIEW: Systemic therapies in neuroendocrine tumors and novel approaches toward personalized medicine. Endocrine-related cancer 2016;23:T135-t154. 12 Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, Hobday TJ, Okusaka T, Capdevila J, de Vries EG, Tomassetti P, Pavel ME, Hoosen S, Haas T, Lincy J, Lebwohl D, Oberg K, Rad001 in Advanced Neuroendocrine Tumors TTSG: Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med 2011;364:514-523. 13 Pavel ME, Hainsworth JD, Baudin E, Peeters M, Horsch D, Winkler RE, Klimovsky J, Lebwohl D, Jehl V, Wolin EM, Oberg K, Van Cutsem E, Yao JC, Group R-S: Everolimus plus octreotide long-acting repeatable for the treatment of advanced neuroendocrine tumours associated with carcinoid syndrome (RADIANT-2): a randomised, placebo-controlled, phase 3 study. Lancet 2011;378:2005-2012. 14 Raymond E, Dahan L, Raoul JL, Bang YJ, Borbath I, Lombard-Bohas C, Valle J, Metrakos P, Smith D, Vinik A, Chen JS, Horsch D, Hammel P, Wiedenmann B, Van Cutsem E, Patyna S, Lu DR, Blanckmeister C, Chao R, Ruszniewski P: Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. The New England journal of medicine 2011;364:501-513. 15 Yao JC, Fazio N, Singh S, Buzzoni R, Carnaghi C, Wolin E, Tomasek J, Raderer M, Lahner H, Voi M, Pacaud LB, Rouyrre N, Sachs C, Valle JW, Delle Fave G, Van Cutsem E, Tesselaar M, Shimada Y, Oh DY, Strosberg J, Kulke MH, Pavel ME, Rad001 in Advanced Neuroendocrine Tumours FTSG: Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or
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gastrointestinal tract (RADIANT-4): a randomised, placebo-controlled, phase 3 study. Lancet 2016;387:968-977. 16 Shipkova M, Hesselink DA, Holt DW, Billaud EM, van Gelder T, Kunicki PK, Brunet M, Budde K, Barten MJ, De Simone P, Wieland E, Lopez OM, Masuda S, Seger C, Picard N, Oellerich M, Langman LJ, Wallemacq P, Morris RG, Thompson C, Marquet P: Therapeutic Drug Monitoring of Everolimus: A Consensus Report. Therapeutic drug monitoring 2016;38:143-169. 17 Wolin EM: PI3K/Akt/mTOR pathway inhibitors in the therapy of pancreatic neuroendocrine tumors. Cancer Lett 2013;335:1-8. 18 Zaytseva YY, Valentino JD, Gulhati P, Evers BM: mTOR inhibitors in cancer therapy. Cancer Lett 2012;319:1-7. 19 Briest F, Grabowski P: PI3K-AKT-mTOR-signaling and beyond: the complex network in gastroenteropancreatic neuroendocrine neoplasms. Theranostics 2014;4:336-365. 20 Zatelli MC, Fanciulli G, Malandrino P, Ramundo V, Faggiano A, Colao A: Predictive factors of response to mTOR inhibitors in neuroendocrine tumours. Endocrine-related cancer 2016;23:R173-183. 21 Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, Ruan DT, Hanna GJ, Haddad RI, Getz G, Kwiatkowski DJ, Carter SL, Sabatini DM, Janne PA, Garraway LA, Lorch JH: Response and acquired resistance to everolimus in anaplastic thyroid cancer. The New England journal of medicine 2014;371:1426-1433. 22 He K, Chen D, Ruan H, Li X, Tong J, Xu X, Zhang L, Yu J: BRAFV600E-dependent Mcl-1 stabilization leads to everolimus resistance in colon cancer cells. Oncotarget 2016 23 Vandamme T, Beyens M, de Beeck KO, Dogan F, van Koetsveld PM, Pauwels P, Mortier G, Vangestel C, de Herder W, Van Camp G, Peeters M, Hofland LJ: Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors. Br J Cancer 2016;114:650-658. 24 Capozzi M, Caterina I, De Divitiis C, von Arx C, Maiolino P, Tatangelo F, Cavalcanti E, Di Girolamo E, Iaffaioli RV, Scala S, Tafuto S: Everolimus and pancreatic neuroendocrine tumors (PNETs): Activity, resistance and how to overcome it. International journal of surgery (London, England) 2015;21 Suppl 1:S89-94. 25 Zitzmann K, Ruden J, Brand S, Goke B, Lichtl J, Spottl G, Auernhammer CJ: Compensatory activation of Akt in response to mTOR and Raf inhibitors - a rationale for dual-targeted therapy approaches in neuroendocrine tumor disease. Cancer Lett 2010;295:100-109. 26 Iida S, Miki Y, Ono K, Akahira J, Nakamura Y, Suzuki T, Sasano H: Synergistic anti-tumor effects of RAD001 with MEK inhibitors in neuroendocrine tumors: a potential mechanism of therapeutic limitation of mTOR inhibitor. Molecular and cellular endocrinology 2012;350:99-106. 27 Valentino JD, Li J, Zaytseva YY, Mustain WC, Elliott VA, Kim JT, Harris JW, Campbell K, Weiss H, Wang C, Song J, Anthony L, Townsend CM, Jr., Evers BM: Cotargeting the PI3K and RAS pathways for the treatment of neuroendocrine tumors. Clinical cancer research : an official journal of the American Association for Cancer Research 2014;20:1212-1222. 28 Capurso G, Fendrich V, Rinzivillo M, Panzuto F, Bartsch DK, Delle Fave G: Novel molecular targets for the treatment of gastroenteropancreatic endocrine tumors: answers and unsolved problems. International journal of molecular sciences 2012;14:30-45. 29 Aristizabal Prada ET, Nolting S, Spoettl G, Maurer J, Auernhammer CJ: The Novel Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) Alone and in Dual-Targeting Approaches Demonstrates Antitumoral Efficacy in Neuroendocrine Tumors in vitro. Neuroendocrinology 2017 30 Peyressatre M, Prevel C, Pellerano M, Morris MC: Targeting cyclin-dependent kinases in human cancers: from small molecules to Peptide inhibitors. Cancers 2015;7:179-237. 31 Luo M, He H, Kelley MR, Georgiadis MM: Redox regulation of DNA repair: implications for human health and cancer therapeutic development. Antioxidants & redox signaling 2010;12:1247-1269. 32 Georgakilas AG: Oxidative stress, DNA damage and repair in carcinogenesis: have we established a connection? Cancer Lett 2012;327:3-4.
P a g e | 22
33 Colussi C, Parlanti E, Degan P, Aquilina G, Barnes D, Macpherson P, Karran P, Crescenzi M, Dogliotti E, Bignami M: The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool. Current biology : CB 2002;12:912-918. 34 Fujikawa K, Kamiya H, Yakushiji H, Nakabeppu Y, Kasai H: Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP. Nucleic acids research 2001;29:449-454. 35 Gad H, Koolmeister T, Jemth AS, Eshtad S, Jacques SA, Strom CE, Svensson LM, Schultz N, Lundback T, Einarsdottir BO, Saleh A, Gokturk C, Baranczewski P, Svensson R, Berntsson RP, Gustafsson R, Stromberg K, Sanjiv K, Jacques-Cordonnier MC, Desroses M, Gustavsson AL, Olofsson R, Johansson F, Homan EJ, Loseva O, Brautigam L, Johansson L, Hoglund A, Hagenkort A, Pham T, Altun M, Gaugaz FZ, Vikingsson S, Evers B, Henriksson M, Vallin KS, Wallner OA, Hammarstrom LG, Wiita E, Almlof I, Kalderen C, Axelsson H, Djureinovic T, Puigvert JC, Haggblad M, Jeppsson F, Martens U, Lundin C, Lundgren B, Granelli I, Jensen AJ, Artursson P, Nilsson JA, Stenmark P, Scobie M, Berglund UW, Helleday T: MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature 2014;508:215-221. 36 Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Gokturk C, Sanjiv K, Stromberg K, Pham T, Berglund UW, Colinge J, Bennett KL, Loizou JI, Helleday T, Knapp S, Superti-Furga G: Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature 2014;508:222-227. 37 Kawamura T, Kawatani M, Muroi M, Kondoh Y, Futamura Y, Aono H, Tanaka M, Honda K, Osada H: Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival. Scientific reports 2016;6:26521. 38 Petrocchi A, Leo E, Reyna NJ, Hamilton MM, Shi X, Parker CA, Mseeh F, Bardenhagen JP, Leonard P, Cross JB, Huang S, Jiang Y, Cardozo M, Draetta G, Marszalek JR, Toniatti C, Jones P, Lewis RT: Identification of potent and selective MTH1 inhibitors. Bioorganic & medicinal chemistry letters 2016;26:1503-1507. 39 Kettle JG, Alwan H, Bista M, Breed J, Davies NL, Eckersley K, Fillery S, Foote KM, Goodwin L, Jones DR, Kack H, Lau A, Nissink JW, Read J, Scott JS, Taylor B, Walker G, Wissler L, Wylot M: Potent and Selective Inhibitors of MTH1 Probe Its Role in Cancer Cell Survival. Journal of medicinal chemistry 2016;59:2346-2361. 40 Aristizabal Prada ET, Orth M, Nolting S, Spottl G, Maurer J, Auernhammer C: The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS One 2017;12:e0178375. 41 Bernier J, Hall EJ, Giaccia A: Radiation oncology: a century of achievements. Nature reviews Cancer 2004;4:737-747.